# A multi-analyte liquid biopsy approach for nonseminomatous testicular germ cell tumors: combining cfDNA and N-glycan analysis in blood and seminal plasma

## TL;DR

This study introduces a new blood and seminal plasma test combining DNA and sugar molecule analysis to improve early detection of a type of testicular cancer that is hard to diagnose.

## Contribution

A novel multi-analyte liquid biopsy approach combining cfDNA and N-glycan profiling for improved diagnostics of nonseminomatous testicular germ cell tumors.

## Key findings

- Combined cfDNA and N-glycan profiling in blood plasma achieved an AUC of 0.96 and identified 85% of STM-negative patients.
- LINE-1 cfDNA methylation levels returned to normal post-surgery, indicating potential for treatment monitoring.
- Seminal plasma analysis showed an AUC of 0.83 and identified 85% of STM-negative patients.

## Abstract

Nonseminomatous testicular germ cell tumors (NSE) present significant diagnostic challenges, especially for the early detection of serum tumor marker (STM) negative cases. Current diagnostic tools are limited, highlighting the need for innovative approaches. This study investigates a novel multi-analyte approach combining circulating cell-free DNA (cfDNA) and N-glycan profiling in both blood and seminal plasma to improve NSE diagnostics.

The study included 41 NSE patients and 114 healthy controls. Diagnostic potential of cfDNA parameters (quality and fragmentation), cfDNA methylation (RASSF1A, PRSS21, and LINE-1) and N-glycan alterations in blood plasma and seminal plasma samples was investigated using logistic regression models. Pre- and post-radical orchidectomy longitudinal samples from NSE patients were analyzed to assess surgical treatment response and disease monitoring utility.

Blood plasma analysis of combined cfDNA and N-glycan profiling demonstrated high diagnostic precision, with an AUC of 0.96, identifying 85% of STM-negative patients and all pure-form teratomas. Post-operative blood plasma analysis showed that LINE-1 cfDNA methylation levels returned to those of healthy controls. Seminal plasma analysis revealed an increased cfDNA fragmentation index (CFI) and cfDNA methylation changes in LINE-1 and PRSS21, with an AUC of 0.83 and identifying 85% of STM-negative patients.

The proposed multi-analyte approach significantly improves early diagnostics of NSE, particularly for STM-negative cases and teratomas. LINE-1 cfDNA methylation is a promising biomarker for NSE detection and treatment monitoring. These findings could transform diagnostic strategies and patient management in testicular germ cell tumors, with potential applications in reducing overtreatment and improving outcomes .

The online version contains supplementary material available at 10.1186/s12935-025-03887-8.